Babesia microti, the primary etiologic agent of human babesiosis, is a tick borne eukaryotic apicomplexan pathogen that is an increasing threat to the US public health. Although B. microti is primarily a tick borne pathogen, it can be transmitted by transfusion of infected blood. Despite the well-recognized threat, there is no FDA licensed test to screen donor blood for B. microti. This inability to prevent Babesia contamination of the US blood supply has resulted in transfusion-transmitted Babesia emerging as the leading cause of death due to microbial contamination of US blood supplies. In this phase 1 STTR application, we will focus on developing monoclonal antibodies (mAbs) for a rapid antigen-capture assay that is both sensitive and specific for the detection of B. microti antigens in human blood. This assay will fill the current gap in the ability to protect the US blood supply against B. microti-infected blood. Very few antigenic peptides have been identified in B. microti that could be used in an antigen capture assay. Consequently, in Aim 1 we will use a novel procedure to generate mAbs to the full spectrum of Babesia antigens generated during infection and identify peptides recognized by the mAbs. In Aim 2 we will express these Babesia antigens in an innovative Toxoplasma expression system to retain conformational in addition to linear epitopes in the proteins that may be important for mAb recognition. This expression system will be used to purify a consistent amount of Babesia antigens for use in downstream assay-development and as standards for our prototype assay. In Aim 3 we will identify non-interfering pairs of monoclonal antibodies to be used as a capture and reporter for each antigen to generate a prototype antigen capture assay for Babesia.